Despite advances in anti-platelet and anti-thrombotic treatment regimens, cardiovascular diseases remain the leading cause of death in the United States. Clinically employed anti-platelet and anti-thrombotic agents include heparin, aspirin, integrilin, and anti-GP IIb/IIIa antibodies (c7E3 Fab, abciximab, or ReoPro). α-Thrombin, generated at the site of vessel injury, is generally assumed to catalyze the hydrolysis of an N-terminal peptide from the human platelet 7-transmembrane thrombin receptor, protease activated receptor 1 (PAR-1), which initiates a cascade of molecular reactions leading to thrombus formation. Thrombin-induced activation of PAR-1, as for other agonist-activated platelet receptors, results in an outside-in signal transduced process followed by the alteration of the surface integrin, GP IIb/IIIa, by an inside-out signal (Schwartz, M. A. et al. 1995. Ann. Rev. Cell Biol. 11:549-599). The conformational change of GP IIb/IIIa leads to the Ca+2-dependent binding of the bi-functional fibrinogen molecule (Bodary, S. C. et al. 1989. J. Biol. Chem. 264:18859-18862). The fibrinogen-GP IIb/IIIa binding sites recognize RGDX sequences in the fibrinogen α chains and an LGGAKQAGDV sequence on the γ chains (Bennett, J. S. et al. 1988. J. Biol. Chem. 263:12948-12953). Potential competing peptides of RGDS and peptides including the γ sequence LGGAKQAGDV were found to be effective antagonists of platelet aggregation (Hawiger, J. et al. 1989. Biochemistry 28:2909-2914). Anti-GP IIb/IIIa antibodies such as c7E3 Fab (Coller, B. S. 1997. J. Clin. Invest. 99:1467-1471; Coller, B. S. 1997. Thromb. Haemost. 78:730-735) and LJ-CP8 (Niiya, K. et al. 1987. Blood 70:475-483) are also potent inhibitors of fibrinogen binding to this glycoprotein complex in activated platelets.
Early studies of the cellular thrombin receptor indicated that more than one species exists in platelets (Greco, N. J. and G. A. Jamieson. 1991. PSEBM 198:792-799; Harmon, J. T. and G. A. Jamieson. 1986. J. Biol. Chem. 261:15928-15933). The first cellular thrombin receptor cloned and sequenced was PAR-1 (Vu, T-K. H. et al. 1991. Cell 64:1057-1068). Human platelets respond to PAR-1 and a second minor receptor PAR-4 (Kahn, M. L. et al. 1998. Nature 394:690-694) while the recently cloned PAR-3 is either absent on human platelets or present in trace amounts (Ishihara, H. et al. 1997. Nature 386:502-506). Mouse platelets respond to α-thrombin primarily through PAR-3 and secondarily through PAR4, with no involvement of PAR-1 (Vu, T-K. H. et al. 1991. Cell 64:1057-1068). Another platelet membrane protein, GP Ib, may also function at least in part as a thrombin receptor (Greco, N. J. and G. A. Jamieson. 1991. PSEBM 198:792-799; Harmon, J. T. and G. A. Jamieson. 1986. J. Biol. Chem. 261:15928-15933; Clemetson, K. J. 1995. Thromb. Haemost. 74:111-116; Greco, N. J. et al. 1996. Biochemistry 35:915). A major role of GP Ib, complexed with GPIX, is the specific interaction with subendothelium-bound von Willebrand factor (vWF) under high shear rates to facilitate platelet adhesion to injured vascular walls (Ruggeri, Z. M. 1994. Semin. Hematol. 31:229-239). The expression on the plasma membrane of the vWF receptor, GP Ib, requires the stable expression of GP Ibβ, GP Ibα, and GPIX (Lopez, J. A. et al. 1991. J. Biol. Chem. 267:12851-12859). The GP Ib-IX complex associates with the cytoskeletal actin binding protein (ABP) via the cytoplasmic domain of GP Ibα (Andrews, R. K. and J. E. B. Fox. 1992. J. Biol. Chem. 267:18605-18611; Cunningham, J. G. et al. 1996. J. Biol. Chem. 271:11581-11587). This GP Ibα-ABP association is initiated by the binding of vWF to GP Ib and appears to be linked to vWF-induced transmembrane signaling (Cunningham, J. G. et al. 1996. J. Biol. Chem. 271:11581-11587). Signal transduction appears to be regulated at least in part by one form of the 14-3-3 zeta protein (Du, X. et al. 1996. J. Biol. Chem. 271:7362-7367) and its association with the GP Ib-IX-V complex (Andrews, R. K. et al. 1998. Biochemistry 37:638-647). The GP Ib receptor also possessed a thrombin binding site that may respond to lower concentrations of thrombin than required to activate the PARs (Greco, N. J. and G. A. Jamieson. 1991. PSEBM 198:792-799; Harmon, J. T. and G. A. Jamieson. 1986. J. Biol. Chem. 261:15928-15933). The GP Ib-thrombin complex may prime the activation of PAR-1 as the thrombin levels rise (Greco, N. J. and G. A. Jamieson. 1991. PSEBM 198:792-799; Clemetson, K. J. 1995. Thromb. Haemost. 74:111-116).
The physiologic functions of the three purported platelet thrombin receptors (PAR-1, PAR-4 and GP Ib) have not yet been clearly defined. A functional role for PAR-1 in α-thrombin-induced platelet aggregation has been shown in vitro (Vu, T-K. H. et al. 1991. Cell 64:1057-1068). However, no comparable responses have yet been described for PAR-4 or GP Ib using a natural thrombin agonist.